Polyreactive immunoglobulins and natural antibodies are different substances

It was shown that the polyreactive immunoglobulins of intact animal or human sera and the natural antibodies of these sera have different properties. Polyreactive immunoglobulins interact non-specifically with various antigens and this interaction is strongly dependent on an exposure of hydrophobic sites by antigens and, probably, by polyreactive immunoglobulins. Tween 20 and low temperature can substantially suppress this reaction. Various non-related soluble antigens can inhibit the binding of PRIG to any immobilized denatured antigen with similar efficiency. In contrast, natural antibodies interact specifically with appropriate antigens and this interaction can be suppressed only by the same or serologically similar competing antigens. Intact sera contain appreciable amount of polyreactive immunoglobulins, apparently much higher concentration than the concentration of natural antibodies. Biological functions of polyreactive immunoglobulins still remain unknown.     

Transformation of specific antibodies to polyreactive immunoglobulins and their binding to blood vessel walls

It was shown that 30-50% ethanol or 40-70% dimetilsulfoxide could efficiently induce in vitro transformation of specific monoclonal antibodies (mAbs) into non-specific polyreactive immunoglobulins (PRIG). Intravenous injection 0.4 ml of FeSO4-EDTA mixture (60 and 30 mkM respectively) could induce increase of PRIG reactivity in the blood-stream. Intramuscle injection of either 0.1 ml of 40% ethanol, or 0.1 ml of FeSO4-EDTA mixture into muscle of hind limb of C57B1 mice leads to the substantial binding of circulated immunoglobulins to the blood vessels of the muscle. The similar effect could also be induced by ischemia/reperfusion of mice hind limb. In the case of intravenous injection of specific to ovalbumin biotinilated mAbs, the subsequent intramuscle injection of 0.1 ml of 40% ethanol induces apparent transformation of these mAbs into PRIG and their binding to the blood vessels. Intramuscle injection of 0.1 ml of FeSO4-EDTA mixture induces less than ethanol though noticeable effect. The obtained data have shown that cord-blood circulating specific antibodies could be transformed into PRIG at some conditions in vivo. If so, this process might play an important role in the organism defence against infections but could, probably, facilitate the development of atherosclerosis, cardiac infarct, cerebral stroke or tumors.     

Determination of the binding parameters for a pre-existing antigen-antibody mixture

A new method, which allows to evaluate parameters of interaction between antibodies (or receptors) and an antigen (or ligand) is suggested. The method is based on the use of so-called coordinates of dilution suggested by the author earlier. Representation of the data of the titration curves for the mixtures of antibodies (or receptors) and antigen (or ligand) in these coordinates allows one to determine the affinity of interaction and the concentration of antigen (or ligand), which can reversibly block antibodies (or receptors). Simple formulas, which allow to estimate which part of paratopes or bivalent antibodies is free and which part is blocked by the antigen, depending on dilution of the considered system, are also suggested. Such a method could be useful for characterization of infection and autoimmune processes when the antigen and antibodies circulate together in the bloodstream.     

The problem of prozone in serum antibody titration and its mathematical interpretation

The question of the mechanism of "prozone" creation was considered from the point of view of the concentrations of free and semi-blocked bivalent antibodies in the mixture of these antibodies with monovalent antigen. Using the so called "coordinates of dilution", suggested by the author earlier, it was possible to calculate the relationships between the concentrations of either free or semi-blocked bivalent antibodies and the dilution of the antigen-antibody mixture. It was shown that dilution of antigen-antibody mixture leads to an increase of the concentration of free bivalent antibodies and simultaneous sharp decrease of the concentration of semi-blocked antibodies. It is suggested, that such a relationship is quite enough for the creation of prozone effect in reactions, when only bivalent antibodies are active and semi-blocked antibodies compete with free antibodies, providing inhibition of the reaction.     

Determination of the concentration of ligand-specific molecules, found in mixtures with ligand-nonspecific molecules

A new method, which allows determination of the concentration of ligand-specific molecules in a mixture of these molecules with biochemically similar but ligand-unspecific molecules, is suggested. The method is based on a partial exhaustion of the mixture on a column with immobilized ligand and determination of the part of ligand-specific molecules presented in exhausted mixture. The concentration of monoclonal antibodies specific to bovine serum albumin in a commercial "Sigma" preparation and concentration of polyreactive immunoglobulins in a commercial "Sigma" preparation of bovine immunoglobulins were determined by suggested method.     

ELISA-based method for determining the affinity of bivalent antibodies of two specificities in a mixture

The problem of the evaluation of the affinity for two types of bivalent antibodies in a mixture is considered. It is shown that the binding curve in appropriate coordinates can be used to compose either a system of four equations with four unknowns or a system of two equations with two unknown variables. The numerical solution of these equation systems yields affinity constants for both antibodies and the relationship between concentrations of antibodies studied in the mixture.     

Determining the parameters for receptor-ligand interaction by serial dilution method for the case when the ligand and receptor are in a pre-existing mixture

New methods of determining the binding parameters for ligand-receptor interaction are considered. The considered approaches are based on the earlier suggested method of serial dilution and application of so-called coordinates of dilution. It was shown that the suggested methods allow to evaluate affinity constant and ligand concentration even for the case, when the receptor and corresponding ligand of unknown concentration are in a mixture and their separation from each other is impossible. In this connection the suggested methods are especially useful for studying the ligand-receptor interaction if the receptor is very liable and its purification from the ligand would cause drastic changes of its binding properties.     

Evaluation of intramolecular interaction between complementary domains, connected with a flexible chain

A method for the evaluation of the effective binding parameters for the interaction between two complementary domains connected with a flexible chain is suggested. The calculations are based on the assumption that the chain is absolutely flexible and does not hinder free relative diffusion of the domains in the solution but does not allow the domains to move away from each other further than the length of the chain. Then, if the distance between the connected domains and the affinity of the interaction between disconnected domains are known, the suggested method allows calculation so-called "local concentration" of the domains relative to each other. On this basis, it is possible to estimate the upper limit to the fractional content of domains in complex, which, when constrained by the linking polypeptide chain, may be much higher than implied by the absolute concentrations of the domains.     

Dynamics of the interaction of polyreactive immunoglobulins with immobilized antigens]

The kinetic of polyreactive immunoglobulins (PRIG) and immobilized antigen interaction was examined at different temperatures. It was shown that this process can be described by the so-called "competitive" model, and the relatively simple method for the rate constant determination for this process was developed. According to the "competitive" model PRIG molecule could be either in "active" or in "inactive" state and dynamic equilibrium exist between "active" and "inactive" molecules which strongly depend on incubation temperature. Only "active" PRIG can interact with antigens, and this is the reason of strong temperature dependence of PRIG-antigen interaction. The data also show that the mechanism of PRIG-antigen interaction differ from that of antibody-antigen interaction.